Structure-guided immunogens to elicit pan-coronavirus B cell responses

项目摘要 SARS-CoV-2冠状病毒引起的全球大流行 强调了开发下一代病毒疫苗的必要性，以提供对未来的保护 令人关注的肉瘤病毒和沙门氏病毒。一种方法是实现基于结构的免疫原 设计策略，激发对表面暴露的棘突病毒保守部位的体液反应(S) 糖蛋白。潜在的结合部位包括受体结合域(RBD)和受体结合基序(RBM) 病毒进入所需的。S上其他受保护的遗址，在RBD之外，可能也是两个中和的目标 和非中和响应，提供广泛的CoV保护。项目1将结合计算、结构、 生物物理和单个B细胞分析设计可诱导广谱体液的S和RBD免疫原 对Cov的豁免权。根据对冠状病毒序列的进化分析，单一抗原不太可能 赋予对整个贝塔冠状病毒属足够广泛的免疫力；因此，我们预计将产生一种 S或RBD多价疫苗(含项目3)。最初，我们优先使用CoVS作为ACE2受体 概念验证，但设想我们的设计原则可以扩展到其他新兴的CoV，ACE2 不是受体。施密特实验室将实施结构指导的免疫原设计战略，以 将免疫反应集中在RBM和RBD上的保守表位上。我们将使用异种冠状病毒 RBDS作为分子支架呈现精选的ACE2结合冠状病毒RbM和进一步的免疫焦点 经过改造的葡聚糖可以掩盖非保守的表位。舍尔曼实验室将使用先辈 重建以确定现存冠状病毒为额外的S免疫原。埃勒贝迪实验室将分离出 用来自自然感染、接种疫苗或幼稚的人的单抗(MAbs)来确定CoV的交叉 中和单抗和非中和单抗识别S表面的反应性、保守性表位。我们将定义 单抗交叉反应、交叉中和和效应器功能的广度(与项目3)。《弗里蒙特》 实验室将通过X射线结晶学和低温电子显微镜(Fremont)进行结构分析 实验室)，以确定S的靶向表位；该信息将有助于迭代免疫原 优化以引起对这些新定义的交叉反应表位的响应。项目1的输出将是 优化RBD和S基因，转移到项目3，用于VSV或乍得疫苗平台的疫苗配方。

Project Summary The global pandemic caused by Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) coronavirus (CoV) has highlighted the need to develop next-generation viral vaccines that offer protection against future Sarbecoviruses and Merbecoviruses of concern. One approach is to implement structure-based immunogen design strategies that elicit humoral responses to conserved sites on the surface-exposed spike (S) viral glycoprotein. Potential sites include the receptor binding domain (RBD) and the receptor binding motif (RBM) required for viral entry. Other conserved sites on S, outside the RBD, may also be targets of both neutralizing and non-neutralizing responses offering broad CoV protection. Project 1 will combine computational, structural, biophysical, and single B cell analyses to design S and RBD immunogens that elicit broad-spectrum humoral immunity against CoVs. Based on evolutionary analysis of CoV sequences, it is unlikely that a single antigen will confer broad enough immunity against the entire Betacoronavirus genus; we therefore anticipate generating a polyvalent S or RBD containing vaccine (with Project 3). Initially, we prioritize ACE2-receptor using CoVs as a proof-of-concept but envision that our design principles can be extended to other emerging CoVs where ACE2 is not the receptor. The Schmidt laboratory will implement structure-guided immunogen design strategies to focus immune responses towards conserved epitopes on the RBM and RBD. We will use heterologous CoV RBDs as molecular scaffolds to present selected ACE2-binding CoV RBMs and further immune focus using engineered glycans to mask non-conserved epitopes. The Scheuermann laboratory will use ancestral reconstructions to identify extant CoVs as additional S-based immunogens. The Ellebedy laboratory will isolate monoclonal antibodies (mAbs) from naturally infected, vaccinated, or naïve human subjects to define CoV cross- reactive, conserved epitopes on S recognized by neutralizing and non-neutralizing mAbs. We will define the breadth of mAb cross-reactivity, cross-neutralization, and effector functions (with Project 3). The Fremont laboratory will perform structural analyses by X-ray crystallography and cryo-electron microscopy (Fremont laboratory) of the isolated mAbs to define targeted epitopes on S; this information will aid in iterative immunogen optimization to elicit responses to these newly defined, cross-reactive epitopes. The output of Project 1 will be optimized RBD and S genes to transfer to Project 3 for vaccine formulation in VSV or ChAd vaccine platforms.